Characterization of human α4β2-nicotinic acetylcholine receptors stably and heterologously expressed in native nicotinic receptor-null SH-EP1 human epithelial cells
Characterization of human α4β2-nicotinic acetylcholine receptors stably and heterologously expressed in native nicotinic receptor-null SH-EP1 human epithelial cells
复制标题
DOI：
10.1124/mol.64.6.1283
复制
发表时间：
2003-12-01
影响因子：
3.6
通讯作者：
Lukas, RJ
中科院分区：
文献类型：
--
作者：
Eaton, JB;Peng, JH;Lukas, RJ
由α4和β2亚基组成的自然表达的烟碱乙酰胆碱受体(α4beta2-nAChR)是大脑中高亲和力尼古丁结合位点的主要形式，与尼古丁奖赏、介导尼古丁胆碱能传递、通过其他化学信息调节信号以及许多神经精神障碍有关。为了建立一个研究人α4β2-nAChR的模型系统，允许进行蛋白质化学、功能、药理和表达调控的研究，将人α4和β2亚基稳定地导入天然nAChR缺失的人上皮细胞系SH-EP1。异源表达的α4beta2-nAChR参与H-3标记的表巴替丁的高亲和力、特异性结合(H-EBDN；宏观K-D=10 PM；k(ON)=0.74/分钟/NM，k(OFF)=0.013/分钟)。免疫荧光研究表明，几乎每个转基因细胞中都有α4和β2亚单位蛋白的表达，显微放射自显影研究表明，大多数细胞中都有I-125标记的碘-去氯表巴替丁结合部位的表达。H-EBDN结合竞争研究表明，对尼古丁激动剂的亲和力高，对尼古丁拮抗剂的亲和力低。使用RB-86(+)外排试验进行的异源表达的alpha4beta2-nAChR功能研究表明，表巴替丁、尼古丁和乙酰胆碱完全有效；1，1-二甲基-4-苯基-哌嗪、胞二磷胆碱和琥珀胆碱部分有效；二氢-β-乙二胺乙二胺、十甲氧基溴铵和甲基去甲乌头碱具有竞争性拮抗作用；甲乙酰胺和七叶碱具有非竞争性拮抗作用；潘库溴铵、六甲基溴铵和D-卡巴卡林有混合拮抗作用。这些结果表明，转基因SH-EP1细胞可作为研究人α4β2-nAChR的模型，也揭示了放射性配基结合药物的表观亲和力与人α4β2-nAChR功能位点之间的复杂关系。
Naturally expressed nicotinic acetylcholine receptors composed of alpha4 and beta2 subunits (alpha4beta2-nAChR) are the predominant form of high affinity nicotine binding site in the brain implicated in nicotine reward, mediation of nicotinic cholinergic transmission, modulation of signaling through other chemical messages, and a number of neuropsychiatric disorders. To develop a model system for studies of human alpha4beta2-nAChR allowing protein chemical, functional, pharmacological, and regulation of expression studies, human alpha4 and beta2 subunits were stably introduced into the native nAChR-null human epithelial cell line SH-EP1. Heterologously expressed alpha4beta2-nAChR engage in high-affinity, specific binding of H-3-labeled epibatidine (H-EBDN; macroscopic K-D=10 pM; k(on)=0.74/min/nM, k(off)=0.013/min). Immunofluorescence studies show alpha4 and beta2 subunit protein expression in virtually every transfected cell, and microautoradiographic studies show expression of I-125-labeled iodo-deschloroepibatidine binding sites in most cells. H-EBDN binding competition studies reveal high affinity for nicotinic agonists and lower affinity for nicotinic antagonists. Heterologously expressed alpha4beta2-nAChR functional studies using Rb-86(+) efflux assays indicate full efficacy of epibatidine, nicotine, and acetylcholine; partial efficacy for 1,1-dimethyl-4-phenyl-piperazinium, cytisine, and suberyldicholine; competitive antagonism by dihydro-beta-erythroidine, decamethonium, and methyllycaconitine; noncompetitive antagonism by mecamylamine and eserine; and mixed antagonism by pancuronium, hexamethonium, and d-tubocurarine. These results demonstrate utility of transfected SH-EP1 cells as models for studies of human alpha4beta2-nAChR, and they also reveal complex relationships between apparent affinities of drugs for radioligand binding and functional sites on human alpha4beta2-nAChR.